Cerebral ischemia offers a target for studying the action of biologically and therapeutically relevant cDNAs. The overall aim of this project is to employ an intra-arterially administered HSV amplicon vector for the delivery of the murine endothelial nitric oxide synthase (eNOS) cDNA into the cerebral endothelial cells of mice exposed to an ischemic insult. To accomplish this objective, further understanding is needed of the mechanisms that limit/impede efficient gene delivery in vivo and that produce unwanted viral vector toxicity. Because intra-arterially administered HSV vectors activate the complement cascade in mice and rats, initial vector infection of endothelial cells in cerebral vasculature is impeded and toxicity from subsequent inflammatory reactions occurs. To address these issues, our specific aims are to: 1) Determine the effect of inhibitors of complement activation on HSV amplicon infection of endothelial cells in mouse models of cerebral ischemia, 2) Determine if addition to the amplicon envelope of HSV's glycoprotein C, known to evade complement, will increase the efficiency and safety of gene transfer, 3) Determine if evasion of neutralizing immunity to the HSV amplicon will increase the efficiency and the safety of gene transfer and, 4) Determine if endothelial nitric oxide synthase (eNOS) gene transfect protects mice from cerebral ischemia. We will employ an oversized and ICP27-deleted "helper" HSV cloned in a bacterial artificial chromosome to package the amplicon vector because, unlike other packaging methods, it does not regenerate cytotoxic replication-competent helper HSV. Amplicons expressing reporter genes will be delivered into the cerebral vasculature of mice before and after ischemia and transgene delivery into endothelial cells will be assayed in the presence or absence of transient complement depletion. Amplicons whose envelope posses glycoproteins, shown to allow evasion of complement and immunoglobulins, will also be engineered and tested. Mice with genetic defects in complement function will further elucidate the contribution of these humoral responses in limiting amplicon-mediated transgene expression. These results will provide the basis for exploring whether eNOS cDNA delivery into mouse cerebral endothelium provides a neuroprotective effect in the presence of ischemia and whether eNOS gen transfer an be combined with other pharmacologic methods for raising eNOS activity to achieve additive or supra-additive neuroprotective effects. The significance of these studies is that they will increase our understanding of the factors responsible for limiting HSV amplicon delivery of transgenes into the brain, they will provide an avenue for reducing vector toxicity and will provide a significant advance in applying eNOS therapy for augmenting the microvascular circulation during cerebral ischemia.